Compositions and methods for producing benzylisoquinoline alkaloids

ABSTRACT

The present invention relates to host cells that produce compounds that are characterized as benzylisoquinolines, as well as select precursors and intermediates thereof. The host cells comprise one, two or more heterologous coding sequences wherein each of the heterologous coding sequences encodes an enzyme involved in the metabolic pathway of a benzylisoquinoline, or its precursors or intermediates from a starting compound. The invention also relates to methods of producing the benzylisoquinoline, as well as select precursors and intermediates thereof by culturing the host cells under culture conditions that promote expression of the enzymes that produce the benzylisoquinoline or precursors or intermediates thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a divisional application of U.S. patent applicationSer. No. 14/961,662, filed Dec. 7, 2015, now U.S. Pat. No. 9,376,696,which is a divisional application of U.S. patent application Ser. No.14/614,484, filed Feb. 5, 2015, now U.S. Pat. No. 9,322,039, which is adivisional application of U.S. patent application Ser. No. 11/875,814,filed Oct. 19, 2007, now U.S. Pat. No. 8,975,063, which claims priorityto U.S. Provisional Application Nos. 60/859,149, filed Nov. 15, 2006,and 60/852,954, filed Oct. 19, 2006, each of which are incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. GM077346awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to compositions and methods for producingbenzylisoquinoline alkaloids (BIAs) or molecules involved in theproduction of BIAs. The compositions comprise host cells comprising atleast one heterologous coding sequence that encodes for an enzyme or itsequivalent that is involved in the BIA synthetic pathway.

Background of the Invention

Alkaloids are a diverse group of nitrogen-containing small moleculesthat are produced in plants, marine organisms, and microorganismsthrough complex biosynthetic pathways. These complex molecules exhibit arange of interesting pharmacological activities and have been used asantimalarials, anticancer agents, analgesics, and in treatment ofparkinsonism, hypertension, and central nervous system disorders.

The benzylisoquinoline alkaloids (BIAs) are a family of alkaloidmolecules with over 2,500 defined structures. The most common BIAscurrently utilized as medicinal compounds are synthesized in the opiumpoppy and include the analgesics codeine and morphine. However, manyintermediates in this pathway that do not accumulate to significantlevels in plants are themselves pharmacologically active as analgesics,antimalarials, anticancer agents, and antimicrobial agents. Even formolecules that accumulate to high levels in plants, it would beadvantageous to eliminate the rigorous extraction and purificationprocedures required to isolate these compounds.

Chemical synthesis of these types of molecules is normally a costly andtime-consuming process, often requiring harsh process conditions,generating toxic waste streams, and resulting in low quantities of thechemicals. In addition, many structures are simply unattainable usingtraditional synthesis methods due to the number of chiral centers andreactive functional groups. Alternatively, the production of BIAs can beachieved at relatively low cost and high yields in a microbial host.This will allow for cost-effective large scale production ofintermediate and end-product BIAs.

The inventors have developed methods and compounds for the production ofcomplex BIAs and their intermediates. Specifically, one can generatethese molecules by expressing cloned and synthetic cDNAs in the hostorganism such that precursor molecules naturally produced in yeast,specifically L-tyrosine, are converted to various BIA intermediates inthese engineered strains through a series of specific reactionscatalyzed by recombinant enzymes. Engineered yeast strains can also beused to convert more complex substrates into value-added BIA moleculesusing similar strategies. The novel technology developed is theproduction of this family of alkaloid molecules in yeast from simpleprecursor molecules and/or more complex substrates using yeast oranother microorganism as a host for the production of these molecules.Various BIA intermediates will be produced in yeast and can be useddirectly fir their pharmacological activities or they can be used asstarting molecules for chemical synthesis modifications to placeadditional functional groups on these backbone molecules to alter theirpharmacological activities. For instance, one important intermediatereticuline is a molecule from which a number of pharmacologically activemolecules such as sanguinarine and codeine can be synthesized. Inaddition, host cells can be engineered to produce non-natural alkaloidderivatives by adding novel enzymatic conversion steps to theheterologous pathway or eliminating steps from the native orheterologous pathway.

Microbial biosynthesis enables green synthesis and the production ofthese molecules without extreme reaction conditions and toxic wastestreams. Furthermore, many intermediates of interest do not accumulatein the native plant hosts, and studies have demonstrated that modifyingexpression of specific genes in this pathway in the native plant hostsin order to direct accumulation of specific intermediates ofteninactivates multiple enzymes in the pathway, prohibiting the rationalengineering of plant strains to accumulate specific intermediates.Microbial biosynthesis also eliminates the need for rigorous extractionand purification procedures required to isolate target molecules fromthe native host.

SUMMARY OF THE INVENTION

The present invention relates to host cells that produce compoundsclassified as benzylisoquinoline alkaloids, as well as select precursorsand intermediates thereof. The host cells comprise one, two or moreheterologous coding sequences wherein each of the heterologous codingsequences encodes an enzyme involved in the metabolic pathway of abenzylisoquinoline, or its precursors or intermediates from a startingcompound. The invention also relates to methods of producing thebenzylisoquinoline, as well as select precursors and intermediatesthereof by culturing the host cells under conditions that promoteexpression and activity of the necessary enzymes that produce thebenzylisoquinoline or precursors or intermediates thereof, as well asoptimize the growth rate of the host.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a synthetic pathway present in the host cells of thepresent invention. The pathway begins with tyrosine and ends withreticuline. The pathway can include fewer enzymes than those displayedif the desired end result is one of the intermediates in thetyrosine→reticuline pathway.

FIG. 2 depicts measurement of dopamine production from a culture of hostcells of the present invention.

FIG. 3 depicts alternative pathways for 4-HPA production-from tyrosinethrough either tyramine or 4-hydroxyphenylpyruvate.

FIGS. 4A and 4B depict measurement of norcoclaurine production from aculture of host cells of the present invention. In this particularculture, the cells possessed the NCS heterologous coding sequence andthe growth media was supplemented with dopamine and 4-HPA. FIG. 4A showsthe pathway of norcoclaurine production and FIG. 4B shows thechromatogram confirming production of norcoclaurine.

FIG. 5 depicts the synthetic pathway present in embodiments of hostcells in the present invention. The pathway begins with norlaudanosolineand ends with reticuline. The pathway can include fewer enzymes thanthose displayed if the desired end result is one of the intermediates inthe norlaudanosoline-reticuline pathway.

FIGS. 6A and 6B depict the measurement of intermediates in thenorlaudanosoline→reticuline pathway. FIG. 6A shows the levels of methylnorlaudanosoline produced, and FIG. 6B shows the levels of methyllaudanosoline produced.

FIG. 7 depicts the measurement of reticuline from the host cells shownin FIG. 5.

FIG. 8 depicts the levels of substrate conversion obtained from hostcells expressing different coding sequences available for the enzymes ofthe pathway. The data demonstrate that any combination of enzymevariants (obtained from different native host sources) will producereticuline from the substrate. However, it is observed that certaincombinations produce higher levels of reticuline than others.

FIG. 9 depicts measurement of levels of reticuline production when fedvarious amounts of the starting substrate.

FIG. 10 depicts measurement of levels of reticuline production from thesupplied substrate at various points in the growth cycle of the hostcells. The data demonstrate that the cells continue to produce andaccumulate reticuline well into stationary phase, suggesting differentfermentation strategies for maximizing reticuline production.

FIG. 11 depicts measurement of levels of reticuline production when thecoding sequences for the heterologous enzymes are either integrated intothe genome or expressed from plasmids. The data demonstrate thatintegration does not affect the level of accumulation of the desiredBIA, confirming that the enzymes remain functional and expression issufficient when integrated into the host genome.

FIG. 12 depicts the synthetic pathway present in embodiments of hostcells in the present invention. Although the pathway may be longer,starting from tyrosine or norlaudanosoline as shown in other figures,this particular pathway begins with reticuline and ends with eitherlaudanine or canadine. The pathway can include fewer enzymes than thosedisplayed if the desired end result is one of the intermediates in thenorlaudanosoline-canadine pathway.

FIGS. 13A-13D depict the measurement of intermediates in thereticuline→canadine pathway. FIG. 13A shows the level of canadineproduced, FIG. 13B shows the level of tetrahydrocolumbamine produced,FIG. 13C shows the level of scoulerine produced, and FIG. 13D shows thelevel of reticuline produced. Characteristic MS/MS fragmentationpatterns are also shown for each ion.

FIG. 14 depicts a synthetic pathway from reticuline to thebaine. Notethat the conversion of salutaridinol-7-O-acetate to thebaine isspontaneous, thus not requiring additional enzymatic steps.

FIG. 15 depicts salutaridine production in host cells comprisingheterologous sequences coding for 6OMT, CNMT, 4′OMT, yCPR1 and yCYP2D6.The pathway synthesizes salutaridine when the cells are fedlaudanosoline. The characteristic MS/MS fragmentation pattern is alsoshown for this ion.

FIGS. 16A-16J depict exemplary combination and subcombination pathwaysof the present invention. FIGS. 16A-16C depict exemplary overallcombination pathways and FIGS. 16D-16I depict the subcombinationpathways including the chemical species and enzymes involved in thepathways. Pathway designations in FIGS. 16D-16I refer to the pathwaydesignations in FIGS. 16A-16C. FIG. 16J is but one embodiment of themethods of the present invention that combines a few of thesubcombination pathways to produce a BIA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for producingbenzylisoquinoline alkaloids (BIAS). In particular, the inventionrelates to host cells that have been genetically engineered to expressrecombinant and/or have altered expression of endogenous enzymesinvolved in the biosynthesis of BIAs and their intermediates andderivatives.

In one embodiment, the cells of the present invention are non-plantcells. In a more particular embodiment, the cells are insect cells,mammalian cells, bacterial cells or yeast cells. Representative examplesof appropriate hosts include, but are not limited to, bacterial cells,such as Bacillus subtilis, Escherichia coli, Streptomyces and Salmonellatyphimurium cells and insect cells such as Drosophila S2 and SpodopteraSf9 cells. In one specific embodiment, the cells are yeast cells or E.coli cells. In a more specific embodiment, the yeast cells can be of thespecies Saccharomyces cerevisiae (S. cerevisiae). Yeast is also an idealhost cell because cytochrome P450 proteins, which are involved incertain steps in the synthetic pathways, are able to fold properly intothe endoplasmic reticulum membrane so that activity is maintained, asopposed to bacterial cells which lack such intracellular compartments.Examples of yeast strains that can be used in the invention include, butare not limited to, S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972,SK1 and FL100. In specific examples, the yeast strain is any of S288C(MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATa; his3Δ1;leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0),BY4743 (MATa/MATα; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15;LYS2/lys2Δ0; ura3Δ0/ura3Δ0), and WAT11 or W(R), derivatives of theW303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR;cyr+) which express the Arabidopsis thaliana NADPH-P450 reductase ATR1and the yeast NADPH-P450 reductase CPR1, respectively. In anotherspecific embodiment, the particular strain of yeast cell is W303α (MATα;his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1), which is commerciallyavailable. The identity and genotype of additional examples of yeaststrains can be found at EUROSCARF, available through the World Wide Webat web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html.

Other example of cells that can serve as host cells are included, butnot limited to, the strains listed in the table below.

TABLE I EUROSCARF/Open Biosystems CSY Accession # ORF deleted GeneStrain Background 3 n/a wild type W303; Mat α; his3-11,15 trp1-1 leu2-3ura3-1 ade2-1 142 n/a YER073w/YPL061w ALD5/ALD6 W303; Mat α; his3-11,15trp1-1 leu2-3 ura3-1 ade2-1 152 Y10753 YMR170c ALD2 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 153 Y1075.2 YMR169c ALD3 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 154 Y11671 YOR374w ALD4 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 155 Y10213 YER073w ALD5 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 156 Y12767 YPL061w ALD6 BY4742; Mat α;hls3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 157 Y16510 YML110c COQ5 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 158 Y16246 YOL096c COQ3 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 159 Y13675 YDR316w OMS1 BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 160 30701B YGR001c CVDM003-01A BY4742;Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 161 Y11457 YIL064w BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 162 Y15719 YBR271W BY4742; Mat α;his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 163 B0006B YJR129c CEN.EN2-1B BY4742;Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 164 B0199B YNL024c CEN.HE27-2CBY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 165 Y12984 YNL092wBY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 166 Y12811 YPL017cBY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 167 Y12903 YHR209wBY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 417 16236 YOL086C ADH1BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 418 10891 YMR303C ADH2BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 419 16217 YMR083W ADH3BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ure3Δ0; 420 14623 YGL256W ADH4BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 421 13284 YBR145W ADH5BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 422 16460 YMR318C ADH6BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 423 15821 YCR105W ADH7BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; 151 Y10000 wild typeBY4742 Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0

The cells can be in any environment, provided the cells are able toexpress functional heterologous enzymes. In particular, the cells can beused in either in vitro or in vivo experiments. To be clear, in vitro,as used in the present invention, simply means outside of a living cell,regardless of the location of the cell. The term in vivo, on the otherhand, indicates inside a cell, regardless of the location of the cell.In one embodiment, the cells are cultured under conditions that areconducive to enzyme expression and with appropriate substrates availableto allow production of BIAs in vivo. Alternatively, the functionalenzymes can be extracted from the host for production of BIAs under invitro conditions. In another embodiment, the host cells can be placedback into a multicellular host organism. The host cells can be in anyphase of growth, such as, but not limited to, stationary phase andlog-growth phase, etc. In addition, the cultures themselves may becontinuous cultures or they may be batch cultures.

The cell culture conditions for a particular cell type are well-known inthe art and need not be repeated herein. In one particular embodiment,the host cells that comprise the various heterologous coding sequencescan be cultured under standard or readily optimized conditions, withstandard cell culture media and supplements. As one example, standardgrowth media when selective pressure for plasmid maintenance is notrequired may contain 20 g/L yeast extract, 10 g/L peptone, and 20 g/Ldextrose (YPD). Host cells containing plasmids can be grown in syntheticcomplete (SC) media containing 1.7 g/L yeast nitrogen base, 5 g/Lammonium sulfate, and 20 g/L dextrose supplemented with the appropriateamino acids required for growth and selection. Alternative carbonsources which may be useful for inducible enzyme expression includesucrose, raffinose, and galactose. Cells can be grown at 30° C. withshaking at 200 rpm, typically in test tubes or flasks in volumes rangingfrom 1-1000 mL, or larger, in the laboratory. Culture volumes can alsobe scaled up for growth in larger fermentation vessels, for example, aspart of an industrial process.

The term “host cells,” as used in the present invention, are cells thatharbor the heterologous coding sequences of the present invention. Theheterologous coding sequences could be integrated stably into the genomeof the host cells, or the heterologous coding sequences can betransiently inserted into the host cell. As used herein, the term“heterologous coding sequence” is used to indicate any polynucleotidethat codes for, or ultimately codes for, a peptide or protein or itsequivalent amino acid sequence, e.g., an enzyme, that is not normallypresent in the host organism and can be expressed in the host cell underproper conditions. As such, “heterologous coding sequences” includesadditional copies of coding sequences that are normally present in thehost cell, such that the cell is expressing additional copies of acoding sequence that are not normally present in the cells. Theheterologous coding sequences can be RNA or any type thereof, e.g.,mRNA, DNA or any type thereof e.g., cDNA, or a hybrid of RNA/DNA.Examples of coding sequences include, but are not limited to,full-length transcription units that comprise such features as thecoding sequence, introns, promoter regions, 3′-UTRs and enhancerregions.

“Heterologous coding sequences” also includes the coding portion of thepeptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide orenzyme, as well as the coding portion of the Rill-length transcriptionalunit, i.e., the gene comprising introns and owns, as well as “codonoptimized” sequences, truncated sequences or other forms of alteredsequences that code for the enzyme or code for its equivalent amino acidsequence, provided that the equivalent amino acid sequence produces afunctional protein. Such equivalent amino acid sequences can have adeletion of one or more amino acids, with the deletion being N-terminal,C-terminal or internal. Truncated forms are envisioned as long as theyhave the catalytic capability indicated herein. Fusions of two or moreenzymes are also envisioned to facilitate the transfer of metabolites inthe pathway, provided again that catalytic activities are maintained.

Operable fragments, mutants or truncated forms may be identified bymodeling and/or screening. This is made possible by deletion of, forexample, N-terminal, C-terminal or internal regions of the protein in astep-wise fashion, followed by analysis of the resulting derivative withregard to its activity for the desired reaction compared to the originalsequence. If the derivative in question operates in this capacity, it isconsidered to constitute an equivalent derivative of the enzyme proper.

Codon optimization is a well-known technique for optimizing theexpression of heterologous polynucleotides in host cells and is reviewedin Gustafsson, C. et al., Trends Biotechnol, 22:346-353 (2004), which isincorporated by reference in its entirety.

The present invention also relates to heterologous coding sequences thatcode for amino acid sequences that are equivalent to the native aminoacid sequences for the various enzymes. An amino acid sequence that is“equivalent” is defined as an amino acid sequence that is not identicalto the specific amino acid sequence, but rather contains at least someamino acid changes (deletions, substitutions, inversions, insertions,etc.) that do not essentially affect the biological activity of theprotein as compared to a similar activity of the specific amino acidsequence, when used for a desired purpose. The biological activityrefers to, in the example of a decarboxylase, its catalytic activity.Equivalent sequences are also meant to include those which have beenengineered and/or evolved to have properties different from the originalamino acid sequence. Examples of mutable properties include catalyticactivity, substrate specificity, selectivity, stability, solubility,localization, etc. In specific embodiments, an “equivalent” amino acidsequence contains at least 80%-99% identity at the amino acid level tothe specific amino acid sequence, in particular at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in particular, at least95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In somecases, the amino acid sequence may be identical but the DNA sequence isaltered such as to optimize codon usage for the host organism, forexample.

The host cells may also be modified to possess one or more geneticalterations to accommodate the heterologous coding sequences.Alterations of the native host genome include, but are not limited to,modifying the genome to reduce or ablate expression of a specific enzymethat may interfere with the desired pathway. The presence of such nativeenzymes may rapidly convert one of the intermediates or final productsof the pathway into a metabolite or other compound that is not usable inthe desired pathway. Thus, if the activity of the native enzyme werereduced or altogether absent, the produced intermediates would be morereadily available for incorporation into the desired product. Forexample, if the host cell is a yeast cell and the desired pathwayproduces 4-HPA from tyrosine, or a downstream metabolite thereof it maybe beneficial to reduce or ablate expression of the native endogenousalcohol and/or aldehyde dehydrogenase enzymes, which could convert thedesired final product (4-HPA) into tyrosol or 4-hydroxyphenylaceticacid, respectively. Genetic alterations may also include modifying thepromoters of endogenous genes to increase expression and/or introducingadditional copies of endogenous genes. Examples of this include theconstruction/use of strains which overexpress the endogenous yeastNADPH-P450 reductase CPR1 to increase activity of heterologous P450enzymes. In addition, endogenous enzymes such as ARO8, 9, and 10, whichare directly involved in the synthesis of intermediate metabolites, mayalso be overexpressed.

The heterologous coding sequences of the present invention are sequencesthat encode enzymes, either wild-type or equivalent sequences, that arenormally responsible for the production of BIAs in plants. The enzymesfor which the heterologous sequences will code can be any of the enzymesin the BIA pathway, and can be from any known source. For example,Norcoclaurine synthase (NCS; EC 4.2.1.78) is found in at leastThalictrum flavum, Papaver somniferum, and Coptis japonica and is knownto catalyze the condensation reaction of dopamine and4-hydroxyphenylacetaldehyde (4-HPA) to form the trihydroxylated alkaloid(S)-norcoclaurine, which is widely accepted as the first “committed”step in the production of BIAs in plants. The choice and number ofenzymes encoded by the heterologous coding sequences for the particularsynthetic pathway should be chosen based upon the desired product. Forexample, the host cells of the present invention may comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more heterologouscoding sequences.

With regard to the heterologous coding sequences, the sequences are asreported in GENBANK unless otherwise noted. For example, thecodon-optimized CYP2D6 sequence is included for reference along with thehuman monoamine oxidase A sequence with the first 10 amino acidsoptimized to facilitate translation initiation and proper folding inyeast.

A non-exhaustive list of enzymes that are contemplated in the presentinvention is shown in the table below. The host cells of the presentinvention may comprise any combination of the listed enzymes, from anysource. Unless otherwise indicated, Accession numbers in Table I referto GenBank. Some accession numbers refer to the Saccharomyces genomedatabase (SGD) which is available on the world-wide web atwww.yeastgenome.org.

TABLE II Example Source Organism Enzyme Name Abbrev. (Accession #)Reference Catalyzed Reactions L-tyrosine/dopa TYDC1 P. somniferumFacchini, P. J. and De L-tyrosine-→L-tyramine, decaxboxylase 1 U08597Luca,V. J. Biol. L-dopa→L-dopamine T. flavum Chem. 269 (43), AF31415026684-26690 (1994). PUBMED 7929401 L-tyrosine/dopa TYDC2 P. somniferumFacchini, P. J. and De L-tyrosine-→L-tyramine, decarboxylase 2 U08598Luca, V. J. Biol. L-dopa-→L-dopamine V. vinifera Chem. 269 (43),AM429650 26684-26690 (1994). PUBMED 7929401 Cytochrome P450 CYP2D6 H.sapiens Hiroi, T, Imaoka, S, L-tyramine→L-dopamine 2D6 NM000106 andFunae, Y. (R)-reticuline→(R)- S. cerevisiae Biochem Biophys Ressalutaridine codon- Commun. Aug. 28 codeine→morphine optimized 1998;249(3):838-43. PUBMED 9731223 Zhu, W et. Al. The Journal of Immunology.Vol. 1.75, pp. 7357-7362 (2005). NADPH p450 CPR1 S. cerevisiae Turi, T Gand Loper, L-tyramine-→L-dopamine reductase SGDID: J C. J Biol Chem.(R)-reticuline→(R)- 5000001084 Jan. 25, 1992; salutaridine267(3):2046-55. codeine→morphine PUBMED 1730736 Polyphenyloxidase PPO A.bisporus Wichers, H J et. Al. L-tyrosine→L-dopa X85112, Appl. Microbiol.AJ223816 Biotechnol. (2003) 61: 336-341. Tyrosine TyrH R. norvegicusGrima, B., L-tyrosine→L-dopa hydroxylase NM 012740 Lamouroux, A.,Blanot, F., Biguet, N. F. and Mallet, J. Proc. Natl. Acad. Sci. U.S.A.82 (2), 617-621 (1985). PUBMED 2857492 Tyrosine TH2 H. sapiens Grima,B., L-tyrosine→L-dopa hydroxylase NM 000240 Lamouroux, A., Boni, C.,Julien, J. F., Javoy-Agid, F. and Mallet, J. Nature 326 (6114), 707-711(1987). PUBMED 2882428 GTPcyclohydrolase GTPCH1 H. sapiens Leff, S E et.al. Produces BH₄ cofactor I NM 00161 Experimental for tyrosine Neurology151, 249- hydroxylase reaction 64 (1998). L-tyrosine→L-dopa MonoamineMaoA H. sapiens Bach, W J et. al. Proc. L-tyramine→4-HPA oxidase AJ03792 Natl. Acad. Sci. USA. Vol. 85, pp. 4934- 4938, July 1988.Monoamine maoA E. coli Azakami, H et. al. J. L-tyramine→4-HPA oxidaseD2367 Ferment. Bioeng. 77, 315-319, 1994. Tyramine oxidase tynA K.aerogenes Cooper, R A. FEMS L-tyramine→4-HPA AB200269 Microbiol Lett.Jan. 1, 1997; 146(1):85-9. PUBMED 8997710 Aromatic amino ARO8 S.cerevisiae Iraqui, I et. al. Mol. L-tyrosine→4- acid transaminase SGDID:Gen. Genet. (1998) hydroxyphenylpyruvate S000003170 257: 238-248.Aromatic amino ARO9 S. cerevisiae Iraqui, I et. al. Mol. L-tyrosine→4-acid transaminase SGDID: Gen. Genet. (1998) hydroxyphenylpyravateS000001179 257: 238-248. Phenylpyruvate ARO10 S. cerevisiae Vuralhan, Z.et. al. 4-hydroxyphenyl- decarboxylase SGDID: Appl. And Environ.pyruvate→4-HPA S000002788 Microbiol. Vol. 71, No. 6, p. 3276-3284.Norcoclaurine NCS T. flavum, Samanani, N, L-dopamine + 4-HPA→ synthaseAY376412 Liscombe, D K, and (S)-norcoclaurine P. somniferum Facchini, P.The AY860500, Plant Journal (2004) AY860501 40, 302-313. Norcoclaurine6OMT T. flavum Ounaroon, A. et. al (S)-norcoclaurine→ 6-O- AY61057 ThePlant Journal (S)-coclaurine methyltransferase P. somniferum (2003) 36,808-819. norlaudanosoline→ AY268894 6-O-methyl norlaudanosolinelaudanosoline→6-O- methyl laudanosoline Coclaurine-N- CNMT T. flavumChoi, Kum-Boo et. coclaurine→ methyltransferase AY610508 al. J. Biol.Chem. N-methylcoclaurine, P. somniferum Vol. 277, No. 1, pp.laudanosoline→ AY217336 830-835, 2002. N-methyl laudanosoline CytochromeP450 CYP80B1 P. somniferum Pauli, H H and (S)-N- 80B1 AF191772 Kutchan,T M. Plant methylcoclaurine→ J. March 1998; (S)-3′-hydroxy-N-13(6):793-801. methylcoclaurine 4′-O- 4′OMT T. flavum Morishige, T. et.al. 3′-hydroxy-N- methyltransferase AY610510 J. Biol. Chem. Vol.methylcoclaurine→ P. somniferum 275, No. 30, pp. reticuline AY217333,23398-23405, 2000. norlaudanosoline→4′- AY217334 O-methylnorlaudanosoline laudanosoline→4′-O- methyl laudanosoline Berberinebridge BBE P. somniferum Facchini, P. J., (S)-reticuline-→ enzymeAF025430 Penzes, C., (S)-scoulerine Johnson, A. G. and Bull, D. PlantPhysiol. 112 (4), 1669-1677 (1996). PUBMED 8972604 Reticuline 7-O- 7OMTP. somniferum Ounaroon, A. et. al. reticuline→laudaninemethyltransferase AY268893 The Plant Journal (2003) 36, 808-819.Scoulerine 9-O- S9OMT T. flavum Saroanani, N., (S)-scoulerine→(S)-methyltransferase AY610512 Park, S. U. and tetrahydrocolumbamineFacchini, P. J. Plant Cell 17 (3), 915-926 (2005). PUBMED 15722473Canadine CYP719A T. flavum Samanani, N., (S)tetrahydrocolumbamine→synthase AY610513 Park, S. U. and (S)-canadine Facchini, P. J. PlantCell 17 (3), 915-926 (2005). PUBMED 15722473 Ikezawa, N. et. al. J.Biol. Chem. Vol. 278, No. 40, pp. 38557- 38565, 2003. NADPH P450 ATR1 A.thaliana Louerat-Orion B, Reductase partner for reductase NM 118585Perret A, Pompon D. cytochrome P450s Eur J Biochem. Dec. Ex.(S)tetrahydro- 15, 1998; 258(3):1040- columbamine→ 9. (S)-canadineSalutaridine SalR P. somniferum Ziegler, J. et. al. Plantsalutaridine→salutaridinol reductase DQ316261 J. 48 (2), 177-192 (2006)Salutaridinol 7-O- SalAT P. somniferum Grothe, T., Lenz, R.salutaridinol→salutaridinol- acetyltransferase AF339913 and Kutchan, T.M. J. 7-O-acetate→thebaine Biol. Chem. 276 (33), 30717-30723 (2001).PUBMED 11404355 Codeine COR P. somniferum Unterlinner, B.,codeinone→codeine reductase AF108432 Lenz, R. and Kutchan, T. M. PlantJ. 18 (5), 465-475 (1999). PUBMED 1041769 Berbamunine CYP80A1 B.stolonifera Kraus, P F and 2 (R)-N-methylcoclaurine→ synthase U09610Kutchan, T M. Proc guattegaumerine Natl Acad Sci USA.(R)-N-methlcoclaurine + Mar. 14, 1995; (S)-N-methylcoclaurine→92(6):2071-5. berbamunine

In one specific embodiment, the present invention relates to host cellsthat produce 4-Hydroxyphenylacetaldehyde (4-HPA) from tyrosine. Forexample, the host cells that produce 4-HPA from tyrosine comprise atleast two heterologous coding sequences, wherein each of theheterologous coding sequences encodes a separate enzyme that is involvedin the biosynthetic pathway that converts tyrosine to 4-HPA. In a morespecific embodiment, the host cells that produce 4-HPA from tyrosinecomprise L-tyrosine/dopa decarboxylase (TYDC, P. somniferum) and one ofmonoamine oxidase (maoA, E. coli or Homo sapiens) or Tyramine oxidase(tyn A, Klebsiella aerogenes). In another specific embodiment, the hostcells that produce 4-HPA from tyrosine comprise Aromatic amino acidtransaminase (ARO8/ARO9, S. cerevisiae) and Phenylpyruvate decarboxylase(ARO10, S. cerevisiae).

In another specific embodiment, the present invention relates to hostcells that produce dopamine from tyrosine. For example, the host cellsthat produce dopamine comprise at least two heterologous codingsequences, wherein each of the heterologous coding sequences encodes aseparate enzyme that is involved in the biosynthetic pathway thatconverts tyrosine to dopamine. In a more specific embodiment, the hostcells that produce dopamine from tyrosine comprise a Tyrosine/dopadecarboxylase (TYDC, P. sonmiferum) and one of Cytochrome P450 2D6(CYP2D6, H. sapiens) or Codon-Optimized Cytochrome P450 2D6 (CYP2D6, S.cerevisiae). To improve of the activity of CYP2D6, additional copies ofthe yeast NADPH-P450 reductase (yCPR1) may be expressed either from thechromosome or a plasmid. In another specific embodiment, the host cellsthat produce dopamine from tyrosine comprise a Tyrosine hydroxylase (PPOAgaricus bisporus; TH2, H. sapiens; TyrH, Rattus norvegicus) andTyrosine/dopa decarboxylase (TYDC, P. somniferum).

In another specific embodiment, the present invention relates to hostcells that convert tyrosine into norcoclaurine. Regardless of the sourceof the tyrosine starting material, the host cells of the presentinvention that produce norcoclaurine comprise at least threeheterologous coding sequences, wherein each of the heterologous codingsequences encodes a separate enzyme, or its equivalent, that is involvedin the biosynthetic pathway that converts tyrosine to norcoclaurine. Inone specific embodiment, the host cells that produce norcoclaurine fromtyrosine comprise the L-tyrosine/dopa decarboxylase (TYDC, P.somniferum), Monoamine oxidase (MaoA, E. coli), one of Cytochrome P4502D6 (CYP2D6, H. sapiens) or Codon-Optimized Cytochrome P450 2D6 (CYP2D6,S. cerevisiae), and NCS (T. flavum or P. somniferum) coding sequences.To improve the activity of CYP2D6, additional copies of the endogenousyeast P450 NADPH reductase (yCPR1) may be expressed either from thechromosome or a plasmid. Of course, the embodiment above may furthercomprise additional heterologous coding sequences that will continue thesynthetic pathway to create at least one additional metabolite. Forexample, the presence of the heterologous coding sequence that codes forNorcoclaurine 6-O-methyltransferase (6OMT, T. flavum, P. somniferum)will further metabolize norcoclaurine into coclaurine. Other pathwaysto/from norcoclaurine are depicted herein.

The embodiment above can serve as the basis of additional embodiments.For example, embodiments comprising TYDC, CYP2D6, maoA, NCS, 6OMT mayfurther comprise the Coclaurine-N-methyltransferase (CNMT, T. flavum, P.somniferum) heterologous coding sequence, the embodiments of which mayfurther comprise the Cytochrome P450 80B1 (CYP80B1, P. somniferum)heterologous coding sequence, the embodiments of which may furthercomprise the 4′-O-methyltransferase (4′OMT, T. flavum, P. somniferum)heterologous coding sequence, the embodiments of which may furthercomprise Berberine bridge enzyme (BBE, P. somniferum), etc. Theembodiments in which the host cell comprises TYDC, CYP2D6, maoA, NCS,6OMT and CNMT will generate N-methylcoclaurine ultimately from tyrosine.The embodiments in which the host cell comprises TYDC, CYP2D6, maoA,NCS, 6OMT, CNMT and CYP80B1 will generate 3′-Hydroxy-N-methylcoclaurineultimately from tyrosine. The embodiments in which the host cellcomprises TYDC, CYP2D6, maoA, NCS, 6OMT, CNMT CYP80B1 and 4′OMT willgenerate reticuline ultimately from tyrosine. The embodiments in whichthe host cell comprises TYDC, CYP2D6, maoA, NCS, 6OMT, CNMT CYP80B1,4′OMT and BBE will generate scoulerine ultimately from tyrosine. Allstrains containing either CYP2D6 and/or CYP80B1 will likely requireoverexpression of CPR1 and/or ATR1 NADPH-P450 reductases for optimalactivity.

Of course, the desired pathways need not start with tyrosine. Forexample, the synthetic pathways generated in the host cells may startwith laudanosoline, methyl laudanosoline, norlaudanosoline, methylnorlaudanosoline, or another compound that may or may not be normallypresent in the endogenous BIA pathway. Thus, the starting material maybe non-naturally occurring or the starting material may be naturallyoccurring. Additional examples of starting material include, but are notlimited to, tyramine, dopamine, 4-HPA, 4-HPPA, norcoclaurine,coclaurine, N-methylcoclaurine, 3′-hydroxy-N-methylcoclaurine,reticuline, scoulerine, tetrahydrocolumbamine, canadine, laudanine,sanguinarine, morphine, codeine, codeinone and dimethyltetrahydoisoquinoline, e.g.,6,7-dimethyl-1-2-3-4-tetrahydroisoquinoline. Other compounds may also beused as the starting material in the desired synthetic pathway and oneof skill in the art would recognize the necessary starting material,based upon the synthetic pathway present in the host cell. The source ofthe starting material may be from the host cell itself, e.g., tyrosine,or the starting material may be added or supplemented to the host cellfrom an outside source. For example, if the host cells are growing inliquid culture (an in vivo environment), the cell media may besupplemented with the starting material, e.g., tyrosine ornorlaudanosoline, which is transported into the cells and converted intothe desired products.

In one embodiment, the host cells of the claimed invention convertnorlaudanosoline into reticuline. The norlaudanosoline may be generatedthrough a normal or synthetic pathway in the same or different hostcell, or the norlaudanosoline may be fed to the cells from the outside.In this particular embodiment, the host cells comprise 6OMT, CNMT and4′OMT. This embodiment can serve as the basis of additional embodiments.For example, embodiments comprising 6OMT, CNMT and 4′OMT may furthercomprise BBE or Reticuline 7-O-methyltransferase (7OMT, P. somniferum)the embodiment's of which may further comprise Scoulerine9-O-methyltransferase (S9OMT, T. flavum), the embodiments of which mayfurther comprise Canadine synthase (CYP719A, T. flavum). The embodimentsthat comprise 6OMT, CNMT and 4′OMT will generate reticuline fromnorlaudanosoline. The embodiments that comprise 6OMT, CNMT, 4′OMT andBBE will generate scoulerine from norlaudanosoline. The embodiments thatcomprise 6OMT, CNMT, 4′OMT, and CYP2D6 with its reductase partner (CPR1or ATR1) will generate salutaridine from norlaudanosoline. Theembodiments that comprise 6OMT, CNMT, 4′OMT and 7OMT will generatelaudanine from norlaudanosoline. The embodiments that comprise 6OMT,CNMT, 4′OMT, BBE and S9OMT will generate tetrahydrocolumbamine fromnorlaudanosoline. The embodiments that comprise 6OMT, CNMT, 4′OMT, BBE,S9OMT and CYP719A with its reductase partner ATR1 will generate canadinefrom norlaudanosoline.

The following is a non-exhaustive list of exemplary host organismscomprising heterologous coding sequences. The list is not intended tolimit the scope of the invention in any way.

TABLE III Strain Background Plasmid(s) Enzyme(s) CSY73 111/303aP_(ARO9)::TEF, P_(ARO10)::TEF CSY87 W303α pCS251 P_(TEF1)-AbPPO2 CSY88W303α pCS251, P_(TEF1)-AbPPO2, P_(TEF1)-TYDC2 pCS221 CSY94 W303α pCS250,P_(TEF1)-TfNCSΔ10, P_(TEF1)-TYDC2, P_(TEF1)-maoA pCS283 CSY95 W303αChrIV 122460::P_(TEF1)-TYDC2 CSY104 CSY95 ChrV 1100::P_(TEF1)-maoACSY107 W303α pCS222, P_(tetO7)-yCPR1, P_(TEF1)-TYDC2, P_(TEF1)-yCYP2D6pCS330 CSY116 CSY104 his3::P_(GPD)-yCPR1 CSY176 W303αhis3::P_(GAL1)-TfNCS CSY177 W303α his3::P_(GAL1)-Tf6OMT CSY178 W303αhis3::P_(GAL1)-PsNCS2 CSY179 W303α his3::P_(GAL1)-Ps6OMT CSY234 CSY194W(R) pCS330 P_(TEF1)-TYDC2, P_(TEF1)-yCYP2D6 CSY235 CSY194 W(R) pCS222,P_(tetO7)-yCPR1, P_(TEF1)-TYDC2, P_(TEF1)-yCYP2D6 pCS330 CSY307 W303αpCS827, P_(TEF1)-Ps6OMT, P_(TEF1)-PsCNMT, P_(TEF1)-Ps4′OMT pCS830 CSY308W303α pCS828, P_(TEF1)-Ps6OMT, P_(TEF1)-TfCNMT, P_(TEF1)-Ps4′OMT pCS830CSY309 W303α pCS829, P_(TEF1)-Tf6OMT, P_(TEF1)-PsCNMT, P_(TEF1)-Ps4′OMTpCS830 CSY310 W303α pCS772, P_(TEF1)-Tf6OMT, P_(TEF1)-TfCNMT,P_(TEF1)-Ps4′OMT pCS830 CSY311 W303α pCS827, P_(TEF1)-Ps6OMT,P_(TEF1)-PsCNMT, P_(TEF1)-Tf4′OMT pCS831 CSY312 W303α pCS828,P_(TEF1)-Ps6OMT, P_(TEF1)-TfCNMT, P_(TEF1)-Tf4′OMT pCS831 CSY313 W303αpCS829, P_(TEF1)-Tf6OMT, P_(TEF1)-PsCNMT, P_(TEF1)-Tf4′OMT pCS831 CSY314W303α pCS772, P_(TEF1)-Tf6OMT, P_(TEF1)-TfCNMT, P_(TEF1)-Tf4′OMT pCS831CSY288 W303α his3::P_(TEF1)-Ps6OMT, leu2::P_(TEF1)-PsCNMT,ura3::P_(TEF1)-Ps4′OMT CSY334 W303α his3::P_(TEF1)-Ps6OMT,leu2::P_(TEF1)-PsCNMT, ura3::P_(TEF1)-Tf4′OMT CSY316 W303αhis3::P_(GAL1)-Ps6OMT-loxP-KanR, leu2::P_(TEF1)-PsCNMT,ura3::P_(TEF1)-Ps4′OMT CSY317 W303α his3::P_(TEF1)-Ps6OMT,leu2::P_(GAL1)-PsCNMT-loxP-URA3, ura3::P_(TEF1)-Ps4′OMT CSY318 W303αhis3::P_(TEF1)-Ps6OMT, leu2::P_(TEF1)-PsCNMT,ura3::P_(GAL1)-Ps4′OMT-loxP-LEU2 CSY319 W303α his3::P_(TEF1)-Ps6OMT,leu2::P_(TEF1)-PsCNMT, ura3::P_(GAL1)-Tf4′OMT-loxP-LEU2 CSY325 W303αhis3::P_(GAL1)-Ps6OMT-loxP-KanR, leu2::P_(TEF1)-PsCNMT,ura3::P_(TEF1)-Ps4′OMT, gal2::HIS3 CSY326 W303α his3::P_(TEF1)-Ps6OMT,leu2::P_(GAL1)-PsCNMT-loxP-URA3, ura3::P_(TEF1)-Ps4′OMT, gal2::HIS3CSY327 W303α his3::P_(TEF1)-Ps6OMT, leu2::P_(TEF1)-PsCNMT,ura3::P_(GAL1)-Ps4′OMT-loxP-LEU2, gal2::HIS3 CSY328 W303αhis3::P_(TEF1)-Ps6OMT, leu2::P_(TEF1)-PsCNMT,ura3::P_(GAL1)-Tf4′OMT-loxP-LEU2, gal2::HIS3 CSY336 CSY288 pCS1018P_(TEF1)-PsBBE CSY337 CSY288 pCS1070 P_(TEF1)-PsBBE, P_(TEF1)-TfS9OMTCSY338 CSY334 pCS1018 P_(TEF1)-PsBBE CSY339 CSY334 pCS1070P_(TEF1)-PsBBE, P_(TEF1)-Tf59OMT CSY399 pCS1018, pCS1058 P_(TEF1)-PsBBE,P_(TEF1)-TfS9OMT, P_(TEF1)-TfCYP719A, P_(TEF1)-AtATR1 pCS953, CSY288CSY400 pCS1018, pCS1058 P_(TEF1)-PsBBE, P_(TEF1)-TsS9OMT,P_(TEF1)-TfCYP719A, P_(TEF1)-AtATR1 pCS953, CSY334 CSY401 CSY286 pCS1163P_(TEF1)-PsR7OMT CSY402 CSY334 pCS1163 P_(TEF1)-PsR7OMT CSY409 CSY334his3::P_(TEF1)-Ps6OMT, leu2::P_(TEF1)-PsCNMT, ura3::P_(TEF1)-Tf4′OMT,trp1::P_(TEF1)-AtATR1(KanR) CSY410 CSY409 pCS1018, P_(TEF1)-PsBBE,P_(TEF1)-TfS9OMT, P_(TEF1)-TfCYP719A pCS953 CSY424 CSY334 pCS782P_(TEF1)-yCYP2D6 CSY425 CSY409 pCS782 P_(TEF1)-yCYP2D6 CSY426 CSY288trp1::P_(TEF1)-yCPR1(KanR) CSY427 CSY426 pCS782 P_(TEF1)-yCYP2D6

The promoters driving expression of the heterologous coding sequencescan be constitutive promoters or inducible promoters, provided that thepromoters can be active in the host cells. The heterologous codingsequences may be expressed from their native promoters, or non-nativepromoters may be used. Although not a requirement, such promoters shouldbe medium to high strength in the host in which they are used. Promotersmay be regulated or constitutive. In one embodiment, promoters that arenot glucose repressed, or repressed only mildly by the presence ofglucose in the culture medium, should be used. There are numeroussuitable promoters, examples of which include promoters of glycolyticgenes such as the promoter of the B. subtilis tsr gene (encodingfructose biphosphate aldolase) or GAPDH promoter from yeast S.cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase) (BitterG. A., Meth. Enzymol. 152:673 684 (1987)). Other strong promotersinclude the ADHI promoter of baker's yeast (Ruohonen L., et al., J.Biotechnol. 39:193 203 (1995)), the phosphate-starvation inducedpromoters such as the PHO5 promoter of yeast (Hinnen, A., et al., inYeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989),and the alkaline phosphatase promoter from B. licheniformis (Lee. J. W.K., et al., J. Gen. Microbiol. 137:1127 1133 (1991)). Some specificexamples of yeast promoters include inducible promoters such as Gal1-10,Gal1, GalL, GalS, repressible promoter Met25, tetO, and constitutivepromoters such as glyceraldehyde 3-phosphate dehydrogenase promoter(GPD), alcohol dehydrogenase promoter (ADE), translation-elongationfactor-1-alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1),MRP7 promoter, etc. Autonomously replicating yeast expression vectorscontaining promoters inducible by hormones such as glucocorticoids,steroids, and thyroid hormones are also known and include, but are notlimited to, the glucorticoid responsive element (GRE) and thyroidhormone responsive element (TRE). These and other examples are describedU.S. Pat. No. 7,045,290, which is incorporated by reference, includingthe references cited therein. Additional vectors containing constitutiveor inducible promoters such as alpha factor, alcohol oxidase, and PGHmay be used. Additionally any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of genes. Similarly, one of skill in the art can chooseappropriate promoters specific to the host cell, e.g., E. coli. One canalso use promoter selection to optimize transcript, and hence, enzymelevels to maximize production while minimizing energy resources.

Vectors useful in the present invention include vectors for use in yeastand other cells. Yeast vectors can be broken up into 4 generalcategories: integrative vectors (YIp), autonomously replicating highcopy-number vectors (YEp), autonomously replicating low copy-numbervectors (Yap) and vectors for cloning large fragments (YACs). There aremyriad of yeast expression vectors that are commercially available fromsources such as, but not limited to, American Type Culture Collection(ATCC, Manassas, Va., USA) and Invitrogen Corp. (Carlsbad, Calif., USA).

Alternatively, insect cells may be used as host cells. In oneembodiment, the polypeptides of the invention are expressed using abaculovirus expression system (see, Luckow et al., Bio/Technology, 1988,6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Rielly etal. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No.4,879,236, each of which is incorporated herein by reference in itsentirety). In addition, the MAXBAC™ complete baculovirus expressionsystem (Invitrogen) can, for example, be used for production in insectcells.

Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al (MOLECULAR.CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate.Nucleic acid encoding a selectable marker can be introduced into a hostcell on the same vector as that encoding the functional enzyme, or itsequivalent, or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

Similarly, if the host cells are bacterial cells or animal or insectcells, there are a variety of commercially available expression vectorsfrom which to choose. The choice of expression vector system will beapparent to one of skill in the art. One example of a yeast expressionvector includes, but is not limited to, p413-TEF, p426-GPD, pCM190,pRS313, PYES2-NT/A, etc.

The choice of yeast plasmids will depend on the host. For Z. rouxiivectors based on the native cryptic plasmids pSR1 (Toh, E. et al., J.Bacteria 151:1380-1390 (1982)), pSB1, pSB2, pSB3 or pSB4 (Toh-E et al.,J. Gen. Microbiol. 130:2527-2534 (1984)) may be used. Plasmid pSRT303D(Jearnpipatkul, A., et al., Mol. Gen. Genet. 206:88-94 (1987)) is anexample of useful plasmid vector for Zygosaccharomyces yeast.

Methods of transforming yeast for the purposes of the present inventionare well known in the art. Briefly, inserting DNA into yeast can beaccomplished with techniques that include but are not limited to, thoseusing spheroplasts, treating with lithium salts and electroporation. Themethods are used to insert the heterologous coding sequences into thehost cells such that the host cells will functionally express theenzymes or their equivalents and convert the starting/intermediatecompounds into the desired end product.

Of course, the present invention also relates to methods of producingBIAs comprising culturing the host cells under conditions suitable forprotein production such that the heterologous coding sequences areexpressed in the host cells and act upon the starting/intermediatemolecules.

In another embodiment of the present invention the host cells may alsobe used for functional genomics studies in both plant and animals. Forexample, host cells that are able to convert a given substrate such asnorlaudanosoline into reticuline or other downstream BIAs can be used toscreen libraries of plant cDNA sequences to discover enzymes which acton the product molecule. Of course, the screening methods can also beapplied to precursors of BIAs. The screening methods can be accomplishedby cloning a cDNA library from an organism, such as a plant or anyorganism that produces BIAs or an intermediate thereof, e.g., dopamine,into a suitable expression vector, e.g., a yeast expression vector, andtransforming the library of plasmids into the engineered host cells. Thestandard LiAc/SSD/PEG method can be used Single colonies can then begrown in liquid culture in the presence of substrate and the growthmedia or cell extract analyzed by LC-MS. New BIA molecules and thecorresponding enzymes catalyzing their production can be identified bychromatogram peaks not present in strains lacking the cDNA librarysequence. In vitro or other high-throughput methods can also be used ifa suitable assay has been developed for a particular metabolite orbyproduct, for example. An additional area of study where theseengineered host cells can be employed is in the characterization of therecombinant enzymes known or suspected to be involved in these pathways.In particular, host cells expressing one or more heterologous codingsequences can be grown in the presence of various substrates and theresulting metabolites analyzed by LC-MS or other methods. Both in vivoand in vitro methods can be used in this manner to determine thesubstrate specificities of these enzymes and possibly discover newcatalytic activities.

EXAMPLES Example 1—Construction of Yeast Expression Vectors

Standard molecular biology methods were used to construct the yeastexpression vectors. Heterologous coding sequences for the genes ofinterest were received as plasmids, typically suited for expression inE. coli. Coding sequences were either amplified by polymerase chainreaction (PCR) or excised from the vectors if restriction sites werecompatible with the destination vector. Briefly, yeast shuttle vectorswere constructed based on pCM185 and pCM180 which have an ampicillinresistance marker for maintenance in E. coli, URA and TRP selectionmarkers, respectively, and a centromeric (ARS1/CEN4) origin ofreplication for yeast. To construct exemplary vectors, the TEF1 promoterwas amplified from p413-TEF and the CYC1 promoter from pCM190 andassembled with the NCS coding sequence using PCR methods. Primers foreach segment included suitable restriction sites both for cloning intothe plasmid backbone and allowing the coding sequence to be easilyreplaced. This promoter-gene-terminator assembled PCR product was clonedinto XhoI/BamHI sites of pCM185. Similar methods were used to make asecond DNA insert containing the TEF promoter and 6OMT gene, which wasthen cloned into PmeI/NotI sites of the previous vector such that a CYC1terminator for this gene was included on the plasmid backbone. Similarmethods were used to construct the analogous vector with a TRP selectionmarker. In later constructs, the origin of replication was replaced withthe 2μ origin using standard cloning procedures to allow for high copyexpression in yeast. For cloning and expression of desired enzymecombinations, heterologous coding sequences were cloned into theseprimary vectors; restriction sites were changed using site-directedmutagenesis if necessary. To remove the second gene from theseconstructs, vectors were digested with Mini and self-ligated. To removethe first gene from these constructs, the vectors were digested withXhoI and either BamHI or PmeI, ends were blunted using the Klenowenzyme, and self-ligated. Alternatively, a single promoter vector can bemade by cloning the gene of interest between the first promoter andsecond terminator. Analogous vectors with the HIS selection marker werealso constructed as needed; for example, to express more than fourheterologous coding sequences.

Example 2—Production of Codon-Optimized CYP2D6

The coding sequence for CYP2D6 was optimized based on codon usage in S.cerevisiae as well as RNA secondary structure, using commerciallyavailable service providers, such as DNA2.0 Inc. (Menlo Park, Calif.,USA). There are other service providers that offer codon-optimizedsequences, and some algorithms are available on the world-wide web.

The following is an example of a sequence for yeast codon-optimizedCYP2D6 sequence; SalI and NotI restriction sites for cloning areunderlined.

   1 GTCGACATGG CATTGGAAGC ACTAGTCCCT TTAGCTGTAA TTGTAGCAAT   51ATTCCTGTTA TTGGTAGACC TTATGCATAG AAGACAAAGA TGGGCTGCAA  101GATACCCACC CGGCCCACTA CCCTTGCCAG GACTAGGTAA CCTTTTACAT  151GTTGATTTCC AAAATACTCC GTACTGTTTT GATCAATTGA GGAGAAGATT  201CGGAGATGTT TTCAGTCTGC AGTTGGCATG GACACCAGTC GTCGTTTTAA  251ATGGTTTGGC TGCAGTAAGA GAAGCTTTAG TTACGCATGG CGAAGATACG  301GCGGACAGGC CTCCTGTGCC CATTACACAG ATATTGGGTT TCGGACCTAG  351ATCTCAGGGT GTATTCCTTG CCCGTTACGG TCCTGCGTGG AGAGAACAGA  401GAAGGTTTTC TGTATCAACA CTTAGGAATT TGGGTCTAGG CAAGAAATCA  451TTGGAACAAT GGGTGACCGA GGAAGCCGCT TGTTTGTGCG CAGCCTTTGC  501TAATCATTCT GGCCGTCCTT TTAGACCTAA TGGATTACTT GATAAAGCAG  551TATCTAATGT GATTGCCTCC TTAACATGTG GTAGACGTTT TGAGTACGAT  601GACCCAAGGT TTTTGAGATT GTTAGATCTA GCACAAGAGG GATTAAAGGA  651AGAAAGTGGT TTCTTGAGAG AGGTTTTGAA TGCTGTTCCA GTGCTATTAC  701ACATTCCAGC CCTAGCTGGA AAGGTCTTGA GATTTCAAAA GGCTTTCTTA  751ACGCAGCTTG ATGAGTTACT TACAGAGCAT AGGATGACTT GGGATCCTGC  801TCAACCCCCG AGAGATCTAA CCGAGGCCTT CCTGGCTGAA ATGGAAAAAG  851CAAAGGGTAA TCCGGAAAGT TCCTTCAATG ATGAAAACCT GAGAATTGTC  901GTGGCGGACT TGTTCTCTGC CGGAATGGTG ACAACGTCTA CTACTTTGGC  951CTGGGGACTT CTATTAATGA TTCTTCATCC AGACGTCCAG AGAAGAGTGC 1001AACAAGAAAT AGATGATGTG ATAGGACAAG TTAGAAGGCC AGAAATGGGT 1051GACCAGGCAC ATATGCCATA TACGACTGCT GTAATCCATG AAGTGCAACG 1101TTTTGGGGAC ATTGTCCCCT TGGGAATGAC CCACATGACT TCTCGTGATA 1151TTGAAGTACA AGGTTTCAGA ATACCAAAGG GAACTACGCT GATTACGAAT 1201CTGTCTAGCG TGCTAAAAGA CGAAGCTGTC TGGGAGAAGC CATTTAGGTT 1251TCATCCAGAA CACTTCTTAG ACGCTCAGGG TCATTTCGTA AAGCCTGAAG 1301CATTCCTTCC GTTTAGTGCC GGACGTAGGG CGTGTTTGGG TGAACCATTA 1351GCTAGAATGG AATTATTCCT TTTTTTTACA TCTTTATTGC AGCACTTTTC 1401ATTTTCTGTT CCGACTGGCC AACCCAGACC TAGCCATCAT GGTGTTTTTG 1451CTTTCCTAGT TTCTCCCTCT CCTTATGAAT TATGCGCGGT TCCCCGTTGA 1501 GCGGCCGC

The following is an example of human MAO A sequence optimized forexpression in yeast and includes 8 nt preceding the start codon, whichis underlined:

   1 AATTAATAAT GGAAAACCAA GAAAAGGCTT CTATCGCGGG CCACATGTTC   51GACGTAGTCG TGATCGGAGG TGGCATTTCA GGACTATCTG CTGCCAAACT  101CTTGACTGAA TATGGCGTTA GTGTTTTGGT TTTAGAAGCT CGGGACAGGG  151TTGGAGGAAG AACATATACT ATAAGGAATG AGCATGTTGA TTACGTAGAT  201GTTGGTGGAG CTTATGTGGG ACCAACCCAA AACAGAATCT TACGCTTGTC  251TAAGGAGCTG GGCATAGAGA CTTACAAAGT GAATGTCAGT GAGCGTCTCG  301TTCAATATGT CAAGGGGAAA ACATATCCAT TTCGGGGCGC CTTTCCACCA  351GTATGGAATC CCATTGCATA TTTGGATTAC AATAATCTGT GGAGGACAAT  401AGATAACATG GGGAAGGAGA TTCCAACTGA TGCACCCTGG GAGGCTCAAC  451ATGCTGACAA ATGGGACAAA ATGACCATGA AAGAGCTCAT TGACAAAATC  501TGCTGGACAA AGACTGCTAG GCGGTTTGCT TATCTTTTTG TGAATATCAA  551TGTGACCTCT GAGCCTCACG AAGTGTCTGC CCTGTGGTTC TTGTGGTATG  601TGAAGCAGTG CGGGGGCACC ACTCGGATAT TCTCTGTCAC CAATGGTGGC  651CAGGAACGGA AGTTTGTAGG TGGATCTGGT CAAGTGAGCG AACGGATAAT  701GGACCTCCTC GGAGACCAAG TGAAGCTGAA CCATCCTGTC ACTCACGTTG  751ACCAGTCAAG TGACAACATC ATCATAGAGA CGCTGAACCA TGAACATTAT  801GAGTGCAAAT ACGTAATTAA TGCGATCCCT CCGACCTTGA CTGCCAAGAT  851TCACTTCAGA CCAGAGCTTC CAGCAGAGAG AAACCAGTTA ATTCAGCGGC  901TTCCAATGGG AGCTGTCATT AAGTGCATGA TGTATTACAA GGAGGCCTTC  951TGGAAGAAGA AGGATTACTG TGGCTGCATG ATCATTGAAG ATGAAGATGC 1001TCCAATTTCA ATAACCTTGG ATGACACCAA GCCAGATGGG TCACTGCCTG 1051CCATCATGGG CTTCATTCTT GCCCGGAAAG CTGATCGACT TGCTAAGCTA 1101CATAAGGAAA TAAGGAAGAA GAAAATCTGT GAGCTCTATG CCAAAGTGCT 1151GGGATCCCAA GAAGCTTTAC ATCCAGTGCA TTATGAAGAG AAGAACTGGT 1201GTGAGGAGCA GTACTCTGGG GGCTGCTACA CGGCCTACTT CCCTCCTGGG 1251ATCATGACTC AATATGGAAG GGTGATTCGT CAACCCGTGG GCAGGATTTT 1301CTTTGCGGGC ACAGAGACTG CCACAAAGTG GAGCGGCTAC ATGGAAGGGG 1351CAGTTGAGGC TGGAGAACGA GCAGCTAGGG AGGTCTTAAA TGGTCTCGGG 1401AAGGTGACCG AGAAAGATAT CTGGGTACAA GAACCTGAAT CAAAGGACGT 1451TCCAGCGGTA GAAATCACCC ACACCTTCTG GGAAAGGAAC CTGCCCTCTG 1501TTTCTGGCCT GCTGAAGATC ATTGGATTTT CCACATCAGT AACTGCCCTG 1551GGGTTTGTGC TGTACAAATA CAAGCTCCTG CCACGGTCTT GA

Example 3—Production of Truncated NCS

The T. flavum NCS sequence, courtesy of Peter Facchini, was that of theN-terminal Δ10 truncation. To construct the fill-length gene, the first30 nucleotides (coding for 10 amino acids) were included in the forwardprimer sequence used for cloning the gene. For other variants, such asthe Δ19 N-terminal truncation, the forward primer was designed toamplify the gene beginning at the 20^(th) amino acid and including anadditional start codon if the new starting amino acid was not amethionine. To compare expression levels qualitatively, we cloned eachvariant into a yeast expression vector containing a V5 epitope tag(pYES2-NT/A, Invitrogen), transformed the plasmids into the wild-typeyeast strain using the standard LiAc/SSD/PEG method (Gietz, R D andWoods, R A. Methods in Enzymology, Vol. 350, pp. 87-96, 2002), andperformed Western blot analysis on the total protein lysates. Thisshowed That the T. flavum NCSΔ10 was the most highly expressed in yeast,consistent with E. coli studies.

Example 4—Measurement of Dopamine Production

Yeast strains were constructed to produce dopamine from tyrosine. Ahigh-copy—TRP plasmid containing TYDC2 and CYP2D6 both between the TEF1promoter and CYC1 terminator was tested in various yeast strains. Thestandard LiAc/SSD/PEG method was used to transform the plasmid(s) intoyeast. The CYP2D6 activity is enhanced as evidenced by an increase indopamine production when the background strain is W(R), whichoverexpresses CPR1 from the chromosome. An additional increase indopamine accumulation is observed when cells are co-transformed with asecond plasmid expressing additional copies of CPR1 from the tetO₇promoter. Alterations to the growth media have also been shown toenhance the activity of P450s in yeast (Jiang, H and Morgan, J A.Biotechnology and Bioengineering, Vol. 85, No 2, pp. 130-7). Mediacontaining 3.4 g/L yeast nitrogen base, 5 g/L casein hydrolysate, and 20g/L glucose was shown to improve dopamine production over standard SCmedia. For measurement of tyramine and dopamine accumulation, the growthmedia can be analyzed directly by LC-MS. Intracellular concentrationscan be estimated by preparation of cell extracts. Briefly, cells arepelleted at 6000 rpm for 5 min at 4° C. and the supernatant carefullyremoved. Using a pipette, pellets of the cell paste are dropped intoliquid nitrogen and a mortar and pestle used to homogenize the cells.Metabolites are extracted with methanol and solids removed bycentrifugation; the liquid is passed through a syringe filter to removeany remaining debris. Appropriate dilutions are made prior to LC-MSanalysis using 20 μL injection volume. Samples were run on an AgilentZORBAX SB-Aq 3×250 mm, 5 μm column with 0.1% acetic acid as solvent Aand methanol as solvent B. A gradient elution is used to separate themetabolites of interest: 0-10 min at 100% A, 10-30 min 0-90% B, 30-35min 90-0% B, followed by a 5 min equilibration at 100% A. Tyrosine,tyramine, dopamine, and L-dopa elute within the first 10 min so that anisocratic elution may be used if analyzing only these and/or similarmetabolites. Following LC separation, metabolites are injected into anAgilent 6320 ion trap MSD for detection. Extracted ion chromatograms areused to identify peaks for selected ions and compared to availablestandards in terms of elution time and MS fingerprint.

Example 5—Measurement of Norcoclaurine Production

Norcoclaurine was produced using both in vivo and in vitro methods. Forin vitro experiments, protocols were based on published work (Samanani,N, Liscombe, D K, and Facchini, P. The Plant Journal, Vol. 40, pp.302-313). Both E. coli and yeast cells expressing NCS variants werelysed with B-PER or Y-PER (Pierce), respectively, and total proteinextracts were used in the assay. In vitro reactions were analyzed byLC-MS. Samples were run on an Agilent ZORBAX SB-Aq 3×250 mm, 5 μm columnwith 0.1% acetic acid as solvent A and methanol as solvent B. A gradientelution is used to separate the metabolites of interest: 0-10 min at100% A, 10-30 min 0-90% B, 30-35 min 90-0% B, followed by a 5 minequilibration at 100% A. Following LC separation, metabolites areinjected into an Agilent 6320 ion trap MSD for detection. Norcoclaurineelutes at 21.2 min using this method. Without a commercially availablestandard, norcoclaurine is confirmed by its characteristic fragmentationpattern. With the ion trap set to perform MS/MS in the 272 ion, themajor fragments of the parent ion of m/z=107 (benzyl) and m/z=161(isoquinoline) were identified. For in vivo experiments, yeast cellsexpressing NCS were supplemented with dopamine (between 10 μM and 1 mM)and 4-HPA (custom synthesized from Biosynthesis, concentrationundetermined). The above method was used to analyze the growth mediadirectly to detect extracellular norcoclaurine accumulation.

Example 6—Production and Measurement of Reticuline and its Intermediates

For production of reticuline from the substrate norlaudanosoline (orlaudanosoline), yeast cells were transformed with plasmids expressingvarious combinations of 6OMT, CNMT, and 4′OMT coding sequences using thestandard LiAc/SSD/PEG method. Yeast cells were grown in SC media lackinguracil and tryptophan for plasmid maintenance. The growth media(SC-URA/-TRP) was supplemented with norlaudanosoline at concentrationsbetween 1-5 mM from a 10 or 20 mM stock solution in water. Cells weregrown in test tubes at 30° C. with shaking at 200 rpm; volumes rangedfrom 1-10 mL and time points were from 8 hrs up to 1 week followingaddition of substrate. Cells (or an aliquot of culture) were pelletedand the supernatant analyzed directly by LC-MS. Samples were run on anAgilent ZORBAX SB-Aq 3×250 mm, 5 μm column with 0.1% acetic acid assolvent A and methanol as solvent B. A gradient elution is used toseparate the metabolites of interest: 0-10 rain at 100% A, 10-30 min0-90% B, 30-35 min 90-0% B, followed by a 5 min equilibration at 100% A.Following LC separation, metabolites are injected into an Agilent 6320ion trap MSD for detection. Reticuline elutes at 23.6 min with thismethod and the correct structure of this metabolite is confirmed byperforming MS/MS on the 330 ion to produce the fragments m/z=136 andm/z=192. Based on the results from plasmid-based expression, the P.somniferum 6OMT and CNMT were selected with either the P. somniferum orT. flavum 4′OMT as the best enzyme combinations, and these sequenceswere integrated into the chromosome using homologous recombination. Inaddition, strains were constructed to test each enzyme individually,typically using a single high-copy plasmid with the TEF promoter drivingexpression of the coding sequence. For the 6OMT activity, the correctproduct, 6-O-methyl norlaudanosoline, was detected by LC-MS whennorlaudanosoline was present in the growth media; in vitro assays basedon published protocols and using yeast lysates were also used to confirmthis activity (Ounaroon, A et. A1. The Plant Journal, Vol. 36, pp.808-19). Yeast cells expressing the CNMT enzyme converted6,7-dimethyl-1,2,3,4-tetrahydroisoquinoline present at 1 mM in thegrowth media to the correct N-methylated product in vivo. Yeast cellsexpressing the 4′OMT enzyme methylated the substrates norlaudanosolineand laudanosoline in vivo. The correct location of the methyl groupaddition to each substrate is confirmed by performing MS/MS on theselected ion in all cases.

Example 7—Production and Measurement of Downstream Metabolites ofReticuline

For production of metabolites beyond reticuline, yeast strains withchromosomal integrations of 6OMT, CNMT, and 4′OMT were used whenpossible. These host cells contained no selection markers, allowing foradditional coding sequences to be introduced on plasmids. For productionof scoulerine, a plasmid expressing BBE was transformed intoreticuline-producing strain(s) using the standard LiAc/SSD/PEG method.For production of tetrahydrocolumbamine, plasmids expressing BBE andS9OMT were cotransformed. For production of canadine, plasmidsexpressing BBE, S9OMT, CYP719A, and ATR1 were contransformed.Construction of a yeast strain to stably express ATR1 along with thereticuline-producing enzymes and transformed with BBE, S9OMT, andCYP719A plasmids showed an increase in CYP719A activity (compared toplasmid-based expression of ATR1) demonstrated by increased conversionof substrate to canadine. Metabolites were detected in the growth mediawhen supplemented with 1 mM or greater norlaudanosoline orlaudanosoline. Samples were run on an Agilent ZORBAX SB-Aq 3×250 mm, 5μm column with 0.1% acetic acid as solvent A and methanol as solvent B.A gradient elution is used to separate the metabolites of interest: 0-10min at 100% A, 10-30 min 0-90% B, 30-35 min 90-0% B, followed by a 5 minequilibration at 100% A. Following LC separation, metabolites areinjected into an Agilent 6320 ion trap MSD for detection. For eachmetabolite in the pathway, MS/MS was performed and the spectra compared.Based on the patterns observed, it can be confirmed that the peakidentified as canadine, for example, has the same molecular structure asits precursor, tetrahydrocolumbamine. For production of salutaridine,the yeast strain stably expressing 6OMT, CNMT, 4′OMT, and ATR1 wastransformed with a plasmid expressing CYP2D6. When the growth media wassupplemented with norlaudanosoline or laudanosoline, salutaridine wasdetected by LC-MS. The elution time of salutaridine is identical to thatof scoulerine as expected although its fragmentation pattern,particularly the 165 ion, indicates that the structure is in the correct(R) conformation based on the reported fragmentation pattern ofsalutaridinol.

What is claimed is:
 1. A method for the production of abenzylisoquinoline alkaloid-product, the method comprising: culturing aplurality of engineered non-plant cells under conditions suitable forprotein production, the plurality of engineered non-plant cellscomprising a first engineered non-plant cell comprising a firstheterologous coding sequence and a second engineered non-plant cellcomprising a second heterologous coding sequence, wherein the first andsecond heterologous coding sequences encode a first and second enzyme,respectively, that are involved in a metabolic pathway that convertstyrosine into the benzylisoquinoline alkaloid product, wherein the firstand second enzymes are operably connected along the metabolic pathway,wherein the first enzyme involved in the metabolic pathway that producesthe benzylisoquinoline alkaloid product catalyzes at least one reactionthat is selected from the group consisting of: Tyrosine to L-DOPA;L-DOPA to Dopamine; 4-hydroxyphenylacetaldehyde and Dopamine toNorcoclaurine; Tyrosine to 4-hydroxyphenylpyruvate; and4-hydroxyphenylpyruvate to 4-hydroxyphenylacetaldehyde, and wherein thesecond enzyme involved in the metabolic pathway that produces thebenzylisoquinoline alkaloid product catalyzes at least one reaction thatis selected from the group consisting of: Norcoclaurine to Coclaurine;Coclaurine to N-Methylcoclaurine; N-Methylcoclaurine to3′-hydroxy-N-methylcoclaurine; 3′-hydroxy-N-methylcoclaurine toReticuline; Norlaudanosoline to 6-O-methyl-norlaudanosoline;6-O-methyl-norlaudanosoline to 6-O-methyl laudanosoline;6-O-methyl-laudanosoline to Reticuline; Reticuline to Laudanine;Reticuline to Scoulerine; Scoulerine to Tetrahydrocolumbamine;Tetrahydrocolumbamine to Canadine; Reticuline to Salutaridine;Salutaridine to Salutaridinol; Salutaridinol toSalutaridinol-7-O-acetate; and Salutaridinol-7-O-acetate to Thebaine,wherein the first enzyme produces a first product selected from thegroup consisting of L-DOPA, Dopamine, Norcoclaurine,4-hydroxyphenylpyruvate, and 4-hydroxyphenylacetaldehyde, and whereinthe first product is acted upon by one or more enzymes to produce asecond product that is along a metabolic pathway to produce thebenzylisoquinoline alkaloid product, wherein the one or more enzymescomprises the second enzyme, and wherein the second product is selectedfrom the group consisting of: Coclaurine, N-Methylcoclaurine3′-hydroxy-N-methylcoclaurine, Reticuline, 6-O-methyl-norlaudanosoline,6-O-methyl-laudanosoline, Laudanine, Scoulerine, Tetrahydrocolumbamine,Canadine, Salutaridine, Salutaridinol, Salutaridinol-7-O-acetate, andThebaine.
 2. The method of claim 1, wherein the one or more enzymesconsists of the second enzyme.
 3. The method of claim 1, wherein thesecond product is the benzylisoquinoline alkaloid product.
 4. The methodof claim 1, wherein the second product is acted upon by a second set ofone or more enzymes to produce the benzylisoquinoline alkaloid product.5. The method of claim 1, wherein the second product is converted to thebenzylisoquinoline alkaloid product.
 6. The method of claim 1, furthercomprising recovering the benzylisoquinoline alkaloid product from thecell culture.
 7. The method of claim 1, wherein the engineered non-plantcell is selected from the group consisting of microbial cells, insectcells, mammalian cells, bacterial cells, and yeast cells.
 8. A methodfor the production of a benzylisoquinoline alkaloid product, the methodcomprising: culturing a plurality of engineered non-plant cells underconditions suitable for protein production, the plurality of engineerednon-plant cells comprising a first engineered non-plant cell comprisinga first heterologous coding sequence, and a second engineered non-plantcell comprising a second and third heterologous coding sequence, whereinthe first, second, and third heterologous coding sequences encode afirst, second, and third enzyme, respectively, that are involved in ametabolic pathway that converts tyrosine into the benzylisoquinolinealkaloid product, wherein the first, second, and third enzymes areoperably connected along the metabolic pathway, wherein the first enzymeinvolved in the metabolic pathway that produces the benzylisoquinolinealkaloid product catalyzes at least one reaction that is selected fromthe group consisting of: Tyrosine to L-DOPA; L-DOPA to Dopamine;4-hydroxyphenylacetaldehyde and Dopamine to Norcoclaurine; Tyrosine to4-hydroxyphenylpyruvate; and 4-hydroxyphenylpyruvate to4-hydroxyphenylacetaldehyde, and wherein each of the second and thirdenzymes involved in the metabolic pathway that produces thebenzylisoquinoline alkaloid product catalyzes at least one reaction thatis selected from the group consisting of: Norcoclaurine to Coclaurine;Coclaurine to N-Methylcoclaurine; N-Methylcoclaurine to3′-hydroxy-N-methylcoclaurine; 3′-hydroxy-N-methylcoclaurine toReticuline; Norlaudanosoline to 6-O-methyl-norlaudanosoline;6-O-methyl-norlaudanosoline to 6-O-methyl-laudanosoline;6-O-methyl-laudanosoline to Reticuline; Reticuline to Laudanine;Reticuline to Scoulerine; Scoulerine to Tetrahydrocolumbamine;Tetrahydrocolumbamine to Canadine; Reticuline to Salutaridine;Salutaridine to Salutaridinol; Salutaridinol toSalutaridinol-7-O-acetate; and Salutaridinol-7-O-acetate to Thebaine,wherein the first enzyme produces a first product selected from thegroup consisting of L-DOPA, Dopamine, Norcoclaurine,4-hydroxyphenylpyruvate, and 4-hydroxyphenylacetaldehyde, wherein thefirst product is acted upon by a first set of one or more enzymes toproduce a second product, wherein the first set of one or more enzymescomprises the second enzyme, and wherein the second product is selectedfrom the group consisting of: Coclaurine N-Methylcoclaurine,3′-hydroxy-N-methylcoclaurine, Reticuline, 6-O-methyl-norlaudanosoline,6-C)-methyl-laudanosoline, Laudanine, Scoulerine, Tetrahydrocolumbamine,Canadine, Salutaridine, Salutaridinol, Salutaridinol-7-O-acetate, andThebaine, and wherein the second product is acted upon by a second setof one or more enzymes to produce a third product that is along ametabolic pathway to produce the benzylisoquinoline alkaloid product,wherein the second set of one or more enzymes comprises the thirdenzyme, and wherein the third product is selected from the groupconsisting of: Coclaurine, N-Methylcoclaurine,3′-hydroxy-N-methylcoclaurine, Reticuline, 6-O-methyl-norlaudanosoline,6-O-methyl-laudanosoline, Laudanine, Scoulerine, Tetrahydrocolumbamine,Canadine, Salutaridine, Salutaridinol, Salutaridinol-7-O-acetate, andThebaine.
 9. The method of claim 8, wherein the first set of one or moreenzymes consists of the second enzyme, the second set of one or moreenzymes consists of the third enzyme, or both.
 10. The method of claim8, wherein the third product is the benzylisoquinoline alkaloid product.11. The method of claim 8, wherein the third product is acted upon by athird set of one or more enzymes to produce the benzylisoquinolinealkaloid product.
 12. The method of claim 8, wherein the third productis converted to the benzylisoquinoline alkaloid product.
 13. The methodof claim 8, further comprising recovering the benzylisoquinolinealkaloid product from the cell culture.
 14. The method of claim 8,wherein the engineered non-plant cell is selected from the groupconsisting of microbial cells, insect cells, mammalian cells, bacterialcells, and yeast cells.